Process for preparing neuraminidase

ABSTRACT

Process for preparing neuraminidase, by using as the bacterial strain the Arthrobacter Sp in a culture containing low concentrations of N-acetyl-neuraminic acid.

This application is a continuation-in-part of international PCTApplication No. PCT/EP92/02868, filed Dec. 11, 1992.

The applicants hereby claim the priority of PCT Application No.PCT/EP92/02868 under 35 U.S.C. §365(c) and 35 U.S.C. §120.

BACKGROUND OF THE INVENTION

The invention relates to an improved process for preparingneuraminidase, an enzyme playing a leading role as a tool for thebiological research, for the studies concerning the cellular facies andfor the enzymatic analysis of glycoproteins.

PRIOR ART

Former experimental works did show that neuraminidase is an exocellularenzyme, very important because it can modify the proteins and hydrolize,more particularly, the residua of sialic acid, localized in the terminalpositions of glycolipids, in particular of glycosphingolipid GDia.

Neuraminidase is widely distributed throughout viruses andmicroorganisms. This enzyme, in fact, is contained in all viruses of themixovirus group and in the bacteria like Arthrobacter sialaphilus,Vibrio choleral, Clostridium welchii (perfringens), Pseudomonasfluorescens, Ps. stuzeri, Ps. pyocyaneus, Lactobacillus bifidus,Pneumococcus and Diphteroid bacilli and Klebsiella aerogenes.

The preparation of neuraminidase starting from said viruses and bacteriarequires, however, complex media and complex operative conditions,leading to a poor enzyme output.

SUMMARY

The Applicant found an improved process allowing an industrialpreparation of neuraminidase; such process comprises the use of abacterial strain having a rapid aerobian growth in a cheap medium, thusobtaining a high yield, without any appreciable presence either oftoxins or of proteolytic enzymes.

Said process is employing, as the bacterial strain, the Arthrobacter Sp,obtained from the modification of the Arthrobacter Urefaciens ATCC 7562,and comprises the following steps:

a) Arthrobacter Ureafaciens ATCC 7562 is grown on a solid agar medium inthe presence of N-acetyl-neuraminic acid and casein hydrolysate, thusobtaining an axenic consisting of a small glossy brilliant colony,herein defined as Arthrobacter Sp;

b) a culture of the above obtained Arthrobacter Sp is grown in a liquidmedium containing NANA, in a concentration comprised between 0.1 and 1%,and casein hydrolysate;

c) once steady state conditions are reached, the culture of ArthrobacterSp. is made to occur under chemostasis conditions, feeding it with afresh medium which contains NANA and is free from casein hydrolysate,thus obtaining the production of a considerable amount of neuraminidase;

d) the thus obtained neuraminidase is recovered from the media andpurified by means of filtration followed by concentration,ultrafiltration, dialysis, and flow through an ion exchange resin. It isworth noting that the presence of NANA is essential for enzyme output.

BRIEF DESCRIPTION OF THE INVENTION

FIG. 1 is a schematic diagram of an apparatus for the practice of theinvention.

FIG. 2 is a graph which shows the growth rate versus time for Example 1.

FIG. 3 is a graph which shows the growth rate versus time for Example 2.

FIG. 4 is a graph which shows the growth rate versus time for Example 3.

FIG. 5 is a graph which shows the growth rate versus time for Example 4.

DETAILED DESCRIPTION OF THE INVENTION

The features and the advantages of the process for the preparation ofneuraminidase, employing as the bacterial strain the Arthrobacter Spaccording to the present invention, will be more widely illustratedduring the following detailed description. The original microorganism,used during the tests, was the Arthrobacter Urefaciens ATCC 7562,purchased from the American Type Culture Collection.

This microorganism was then modified by growth in a solid mediumcontaining casein hydrolysate (0.1%), N-acetyl-neuraminic acid (NANA)and agar (typically at pH comprised between 7.0 and 7.5, and preferably7.3, and at temperatures comprised between 25° and 30° C.). The growthof the microorganism, in the presence of NANA, is performed by means ofseveral passages, thus obtaining an axenic consisting of a small glossybrilliant colony, defined as Arthrobacter Sp. The strain ArthrobacterSp. was deposited on Apr. 11, 1995 as Arthrobacter ureafaciens VR-1,NCIMB 40717 at: "The National Collections of Industrial and MarineBacteria Limited", 23 St Machar Drive, Aberdeen AB2 1RY, Scotland, UKand the deposit was made under the provisions of the Budapest Treaty.

Arthrobacter ureafaciens ATCC 7562 produces on the above solid mediumtiny, round colonies with a characteristic yellow-amber color. After twoor three days of growth on solid medium, the colonies began to deformand produce a waxy polymer-like substance which gives the colony acreamy appearance.

When these colonies are replated they return to the original colonymorphology (small, round) and with increasing incubation times theydevelop into large creamy colonies.

The colony of the Arthrobacter Sp. used in the present invention wasisolated approximately 24 hours after growth on solid medium accordingto step a) and was chosen because it developed precociously as comparedto the rest of the colonies.

Further, when in presence of other colonies, its colony looked largerand showed a more brilliant and intense yellow-amber color. ArthrobacterSp. shows a feature residing in a high output of neuraminidase, when itis grown in a liquid medium in the presence of low concentrations ofNANA (0.1-1%).

It has to be noted that the precursor strain, Arthrobacter UrefaciensATCC 7562, doesn't give rise to any output of neuraminidase, whenexposed to NANA.

The isolated Arthrobacter Sp. also exhibits the followingcharacteristics:

1. Production of neuraminidase isoenzyme active on GDia substrate.

2. Small gram negative rod which undergo rod-sphere-rod morphogenesisduring cultivation in liquid medium.

3. It produces a bright yellow pigment during growth on liquid medium.

4. It grows on glucose, gluconate, fructose and succinate.

5. When grown on glucose, the neuraminidase production is inhibited.

The medium typically used for the growth of the Arthrobacter Spaccording to step b) of the present process is an aqueous solutioncomprising casein hydrolysate, NANA and salts (such as dibasic ammoniumphosphate, potassium (monobasic) phosphate (KH₂ PO₄), sodium chloride,magnesium sulphate, ferrous sulphate). Optionally, yeast extract may bepresent.

According to preferred conditions of the present invention, theconcentration of NANA during step b) is comprised between 0.1 and 1%(g/100 ml), and the concentration of casein hydrolysate is comprisedbetween 0.1 and 0.5% (g/100 ml).

In step c), the concentration of NANA typically varies from 0.05 to 1%.

The culture of Arthrobacter Sp., both under batch conditions such asthose of step b) and under chemostasis conditions such as those of stepc), is carried out under stirring-aeration conditions, at pH valuescomprised between 7.0 and 7.5 and at temperatures comprised between +20°C. and +30° C.

Preferably, the pH is adjusted at 7.3 and the temperature is equal to26° C.

Steady state (stationary phase) conditions represent a growth state inwhich, after the logarithmic growth phase, the rate of cell division andthe one of cell death are in equilibrium, thus a constant concentrationof microorganism is maintained in the vessel. Stationary stateconditions can be simulated under chemostasis conditions, maintaining anequilibrium state between cell doubling μ and wash out rate D, whereinμ=D e dx/dt is 0 [μ represent the specific growth rate (1/x) (dx/dt),wherein x is the concentration of microorganism in vessel and t is time;(D is dilution rate or number of complete volume changes per unittime)].

According to the present process, steady state conditions arespontaneously reached after the logarithmic growth phase at the end ofstep b), then they are maintained under chemostasis conditions, byfeeding the fresh medium into the reaction vessel.

Preferably, steady state conditions are reached at the end of thelogarithmic phase (step b) when the optical density of the cells in thereaction medium reaches values comprised between 1.5 to 4 O.D., andmaintained during step c) by feeding fresh medium into the reactionvessel at a rate so as to maintain the optical density (O.D.) between 3and 5 units.

It is worth noting that according to the present process the maximumneuraminidase output occurs during the steady state of cells growth.According to a typical embodiment of the present invention, in step b)the used medium for the growth of the Arthrobacter Sp. contains thefollowing ingredients in aqueous solution: casein hydrolysate, yeastextract, NANA, dibasic ammonium phosphate, potassium (monobasic)phosphate (KH₂ PO₄), sodium chloride, magnesium sulphate, ferroussulphate (pH 7.3; temperature 26° C.).

By working under such conditions, steady state conditions are reachedafter 40-60 hours of cultivation.

Typically, the fresh medium can be fed into the reactor vessel when thebacterial mass reaches the value of 0.4 g/l.

According to Examples 1-3, as the bacterial mass reaches the value of0.4 g/l, it is added to the culture some fresh medium, containing 0.5g/l of NANA and free from casein hydrolysate, the flow ratecorresponding to a dilution value (number of volume changes per timeunit) equal to 0.13 h⁻¹ (thus completely replacing the the medium ofstep b) in a 7-8 hours period).

By working under such conditions, a considerable amount of neuraminidaseis produced and is accumulated in the broth. According to a particularlypreferred embodiment of the present invention, step b) is carried outusing as the growing medium for Arthrobacter Sp. an aqueous solutioncontaining casein hydrolysate and NANA as the only carbon sources andstep c) is carried out using a fresh medium containing yeast extractother than NANA as the organic nutrients.

Preferably, the medium used in step b) contains casein hydrolysate andNANA both in a 0.5% amount, while the medium used in step c) contains0.5% of NANA and 0.4% of yeast extract.

Both step b) and c) are preferably carried out at pH 7.3, at 26° C. Whenthe process is carried out according to this preferred embodiment, statesteady conditions corresponding to optical density values comprisedbetween 2.0-2.5, more preferably 2.5, are reached during step b) after18-20 hours of cultivation. During step c), the continuous culturingcultivation are started by feeding the above mentioned fresh medium intothe reaction vessel when the optical density fluctuates between 1.4 and2.5, and said fresh medium is fed at a flow rate so as to raise andmaintain the optical density between 4 and 5 O.D, more preferably at 5O.D. units.

The fresh medium was preferably fed at a flow rate comprised between 27to 35 ml/min, which allowed the liquid culturing medium (10 l) containedin the reactor vessel during phase b) to be completely replaced in a 6-7hours period. Higher flow rates generally led to a decrease in enzymeout put, because the wash out rate of cells was higher than celldoubling time, while flow rates less than 27 ml/min increased cellgrowth rate, but decreased enzyme output.

It is worth noting that under these conditions stationary phase of cellgrowth could be maintained indefinitely.

Under such preferred conditions, a surprisingly high neuraminidaseoutput (up to 7000 U/l after 18 hours) is obtained also during step b),before shifting to the continuous culturing conditions, and what is evenmore surprising, the neuraminidase output during step c) reaches11,000-12,000 U/l. Both these values are well higher than those obtainedaccording to the prior art processes.

Further, this high neuraminidase production can be maintained as long asdesired, for period even longer than 220 hours.

The experimental results relating to the present process suggest thatpresence of casein hydrolysate in the growth medium of step a) isimportant to obtain elevated O.D. values.

Further, the Applicants deem that the addition of yeast extract, a richnitrogen source, to the growth medium used for the chemostasiscultivation according to step c), made the microbial cells morequiescent, facilitated reaching steady-state conditions more easily andallowed maintainance of this steady state for extended periods of time.

The used apparatus is illustrated in FIG. 1. It consists of a continuousflow system, traded by the New Brunswick Instrument Co. as MICROGEN-GENII SYSTEM, wherein A is the reactor (or culture container), C is thevessel containing the fresh medium, D is the tank containing the productand B(in) and B(out) are the pumps fit fop respectively feeding themedia to the reactor and for pumping the product from the reactor to thetank D.

The reactor A (14 liters) is inoculated by an initial culture ofArthrobacter Sp; the optical density of the cellular suspension iscomprised between 0.1 and 0.2 OD units. The cellular subdivision beginsafter a lag phase of 5-7 h. The average exponential or logarithmicgrowth phase is reached after 14 h and the doubling time of the cellularsubdivision is 4.6 h.

According to Examples 2 and 3, the steady state conditions areestablished as the cells reach an optical density between 3 and 4 ODunits.

The steady state of the culture is maintained by feeding the freshsterile medium into the reaction flask, the flow rate beingsubstantially equal to 1.8 l/h. The following examples are supplied fopmerely illustrative purposes and do not limit in any case the scope ofthe invention.

EXAMPLES Growth of Arthrobacter Ureafaciens ATCC 7562 in a Solid Mediumin the Presence of Casein Hydrolysate and N-acetyl-neuraminic Acid andIsolation of Arthrobacter Sp.

Arthrobacter Ureafaciens ATCC 7562 was grown on Petri plates, in a solidmedium having the following composition: casein hydrolysate 1.0 g; yeastextract 0.5 g; NANA 1.0 g; (NH₄)₂ PO₄ 2.0 g; KH₂ PO₄ 1.0 g; NaCl 3.0 g;MgSO₄ 0.1 g; FeSO₄ 0.02 g; Agar (DIFCO) 20.0 g; H₂ O 1 liter.

After 24 hours of incubation (pH 7.3; 30° C.) a small glossy brilliantcolony was isolated, designated as Artrhobacter Sp.

The colony of Arthrobacter Sp. developed precociously with respect tothe rest of colonies. When in the presence of other colonies, itappeared as a single isolated colony, larger and with a more developedyellow-amber color in respect of the colonies in its vicinity.

Materials

In Examples 1, 2 and 3, the medium used to grow Arthrobacter Sp. duringstep b) of the present process was an aqueous solution containing caseinhydrolysate and NANA in the amounts indicated for each example, yeastextract (0.05%) and salts: dibasic ammonium phosphate (0.2%), potassium(monobasic) phosphate (KH₂ PO₄) (0.1%), sodium chloride (0.3%),magnesium sulphate (0.1%), ferrous sulphate (0.02%). pH was 7.3 and thetemperature was equal to 26° C.

EXAMPLE 1 Bacterial Growth According to a NANA Concentration Equal to0.1%

The Arthrobacter Sp was made to grow in a medium together with a 0.1%concentration of casein hydrolysate and with a 0.1% of NANA. Once thesteady state level was reached, a fresh medium was fed to the reactor;such medium contained 0.05% of NANA and salts and was free from caseinhydrolysate. The flow rate was 30 ml/minute and the dilution rate(number of volume exchanges per time unit) was equal to 0.13 h⁻¹.

The results of this preparation are recorded on FIG. 2, where it ispossible to note an enzyme output approximately equal to 700 Units/lduring the steady-state (one Enzyme Unit is an amount of enzymesufficient for hydrolizing 1 μmol/minute of NANA, at pH 5.3 and at 37°C., while employing GDia as the substrate). In said figure, line 9represents the optical density (OD), whereas line 2 represents theenzymatic activity (U/L).

EXAMPLE 2 Bacterial Growth When the NANA Concentration is 1% TheArthrobacter Sp is made to grow in a medium containing 0.1% of caseinhydrolyzate and 1% of NANA.

Once the bacterial growth attained the steady state, the reactor was fedwith a fresh medium (containing 0.05% NANA and salts), free from caseinhydrolysate; the flow rate (of the fresh medium) was 30 ml/minute.

The results were recorded on FIG. 3 and show a steady state output ofapproximately 1400 units/l. Line 3 expresses the optical density (OD)and line 4 expresses the enzymatic activity (U/L).

EXAMPLE 3

Bacterial Growth When the NANA Concentration is 0.1%

The Arthrobacter was made to grow in a medium containing 0.5% of caseinhydrolysate and 0.1% of NANA.

Once the steady state of the bacterial growth was reached, the reactorwas fed with a fresh medium containing 0.05% of NANA and salts, freefrom casein hydrolyzate; the flow rate was 30 ml/minute. The results arerecorded on FIG. 4 and show, in correspondence to the steady state, anenzyme output of about 800 Unit/l.

In said figure, line 5 represents the optical density (OD) and line 6represents the enzymatic activity (V/L).

EXAMPLE 4

The Arthrobacter Sp. was made to grow in a liquid aqueous mediumcontaining 0.5% casein hydrolysate; 0.5% NANA and salts: (NH₄)₂ PO₄0.2%; NaCl 0.3%; KH₂ PO₄ 0.1%; MgSO₄.7H₂ O 0.1%; FeSO₄. 7H₂ O 0.002%, at26° C. During cells growth, pH fluctuates and is adjusted to 7.3.

After 18-20 hours the stationary phase (steady state) conditions werereached (O.D.=2.5 units) and an extremely high out put of neuraminidasewas observed (about 7000 U/l after 18 hours).

O.D. slowly decrease from 2.5 and 1.4 and, after 32 hours, cells wereshifted from batch culture to continuous culturing conditions, feedingthe reactor with a fresh medium containing 0.4% yeast extract, 0.5% NANAand salts, at flow rates between 27 and 35 ml/minute.

Under these conditions, 6-7 hours were necessary for changing one vesselvolume, thus completely replacing the initial medium.

This flow rate sustained a constant cell growth rate corresponding toO.D. values comprised between 3 and 5 units.

Under these conditions, an enzyme output comprised between ofapproximately 10,000 and 11,000 U/l was observed (see Table I). Thesesteady state conditions, corresponding to an extremely highneuraminidase production, were maintained in this example for about 10days, but could have been extended indefinitely longer. Table I reportsthe cell density, the enzyme output and the flow rate of fresh mediumduring 180 hours of Arthrocabter Sp. cultivation. FIG. 5 reports thedata relating to the cultivation over a period of about 220 hours.

Ths start of the continuous culturing condiitons are illustrated by thedouble asterisk in the Table or the arrow in FIG. 5. Curve 14 representsthe senzyme in U/ml, curve 12 represents the pH amd curve 10 representsthe O.D. at 660 nm.

According to Table I, neuraminidase production was lower during lagphase and logarithmic phase (470-720 U/l in the first 12 hours). Then,it dramatically increased up to 7000 U/l when the cells growth enteredthe stationary phase. As the stationary phase proceeds, enzyme activitydecreases to 4000 U/l during the 20-30 hours period. Such a decrease isaccompanied by a decrease in O.D., which is common during latestationary phase when cell death rate tends to exceed cell doublingrate. After 32 hours, the fresh medium is pumped into the reactionvessel at increasing flow rate, from 11 to 27 ml/min, and both O.D. andenzyme output increase.

                  TABLE 1                                                         ______________________________________                                        TIME  CELL DENSITY   ENZYME    FLOW RATE                                      (hrs) O.D.           U/l       (ml/min)                                       ______________________________________                                         0    --             --        *                                               3    0.2            --        *                                               6    0.8              380     *                                              12    1.7              940     *                                              18    2.5            7,370     *                                              20    2.1            6,400     *                                              24    2.1            5,900     *                                              28    1.4            4,690     *                                              32    1.4            4,280      11**                                          40    2.1            6,500     19                                             42    2.5            7,800     26                                             50    3.6            9,750     27                                             62    3.9            11,040    27                                             74    4.6            10,100    28                                             140   4.8            11,000    30                                             150   5.0            10,280    35                                             180   6.0            10,400    35                                             ______________________________________                                         *During the first 32 hours the cells are grown in batch culturing             conditions                                                                    **Start of conditions culturing conditions is marked by the arrow in FIG.     5 or double asterisk in Table I.                                         

With regard to the neuraminidase purification, according to a preferredembodiment of step d), the filtraiton is carried out on a membrane ofcellulose acetate having a pore diameter of 0.2 μm; the concentration iseffected through a cellulose acetate membrane showing a molecularcut-off of 10,000 daltons; the dyalisis is effected against a pH 5.3acetate buffer; and the ion exchange resin has a matrix of dextrancross-linked with epichlorohydrin, functionalised with carboxymethylgroups, and with an exclusion molecular weight limit of 2×10⁵, balancedto pH 5.3 by means of an acetate buffer.

Neuraminidase Purification

The media, collected during the preparation hereinabove, is first of allfiltered, in order to separate the microbial cells. This separation iscarried out by employing a Sartorius filter, having Sartocon II modulesand having a filtering membrane made of cellulose acetate and having apore diameter of 0.2 μm. The supernatant fluid is then concentrated in aDC10LA or SP20 filter (both supplied by the Amicon Corp. of the U.S.A).The S-10Y10 cartridge (supplied by Amicon), through which the filtrateis made to flow, consists of cellulose acetate, showing a M.W.(molecular weight) cut-off of 10,000 daltons.

The ultrafiltration is then made to occur by means of a Amicon CH2filter having a S1Y10 cartridge.

While passing through the filter Amicon DC10LA, 220 l of filtrate areapproximately reduced to 3 l and while passing through the CH2 filterthere is a reduction to 400 ml.

The filtrate is then dialyzed against a pH 5.3 acetate buffer (0.02M),according to the following ratio:

5 volumes of buffer per volume of enzyme.

In our case the Applicant employed 2 l of buffer, in order to dialyze400 ml of enzyme.

The liltrate is then made to pass through an ion exchanging resin,employing a CM Sephadex C-50 (Pharmacia) (matrix: dextran cross-linkedwith epichlorohydrin; functional groups: carboxymethyl groups; exclusionMolecular Weight limit of 2×10⁵.) balanced to pH 5.3 by means of asodium acetate buffer (0.02M). After each purification, the ionexchanging column is regenerated in order to remove the residualproteins, employing 4-5 liters of 1M sodium acetate.

Neuraminidasic Activity Test

1. NANA test.

A few solutions of the blank or standard sample, respectively containing5, 10, 15 and 20 μg of NANA in 200 μl of water are treated with 10 μl ofperiodic acid (25 mM in 0.125 N H₂ SO₄). The mixtures are incubated for5 minutes at 60° C. and the excess of periodate is then reduced by meansof 100 μl of sodium arsenite (1.6% in 0.4N HCl). As the yellow colour ofthe released iodine gets lost (2 minutes), there is added 1 ml of 0.01Mthiobarbituric acid (pH 9) and the sample is covered and heated in aboiling water bath for 7.5 minutes.

The coloured solutions are then cooled by means of an ice-water bath andshaken along with 2 ml of an acetate buffer based on acetone (HCl=2.5%).The extinction level is measured (at 552 nm) by means of aspectrophotometer, against an acetate blank.

2. Determination of the enzymatic activity.

The enzymatic activity of the neuraminidase is determined at thesaturation levels of the substrate concentration, corresponding to 5times the K_(m) value.

The released product is measured by employing the colorimetric technique(thiobarbituric acid method). For instance, when the neuraminidaseenzyme and its substrate (a ganglioside like GDia) are incubated for5-10 minutes and the NANA, hydrolyzed from the substrate, is employedfor evaluating the enzymatic activity, the NANA released in the solutionundergoes a series of reactions, in order to determine its actualamount.

Since NANA is also employed for obtaining the culture media of thesteady state wherefrom the filtrate is extracted, a few check reactionsare carried out, in order to ascertain that the measured NANA be the onecoming from the reaction between enzyme and substrate and not the onecoming as a residue from the original culture.

For instance, a few tests are carried out on 3 test-tubes, as reportedhereinbelow. Each of the test-tubes contains the sodium acetate buffer;moreover the first one contains only GDia substrate (S), the second one[blank (B)] contains only enzyme and the third one contains bothsubstrate and enzyme (E).

    ______________________________________                                                 S          B      E                                                  ______________________________________                                        Buffer     150          150    100 μl                                      Substrate   50          --     50 μl                                       Enzyme     --            50    50 μl                                       ______________________________________                                    

In the first test-tube (S) there should be no NANA. Should NANA beretrieved in the second test-tube (B), its spectrophotometric reading issubtracted from the reading obtained from the reaction in test-tubethree (E). The enzyme activity is therefore indirectly measured as theamount of hydrolized NANA per minute at pH 5.3 and at 37° C., using GDiaas the substrate.

The analysis is performed as follows.

The substrate GDia (50 μl) and the neuraminidase enzyme (50 μl) areplaced in a test-tube, along with 50 mM of sodium acetate buffer (pH5.0), and allowed to react for 5 minutes. 100 μl of 0.125N periodic acidHIO₄ (25 mM in H₂ SO₄) are then added and the reaction is allowed toproceed for 5 more minutes in a heated bath (at 60° C.). At this point,100 μl of sodium arsenite (1.6% in 0.4N HCl) and 1 ml of 0.1Mthiobarbituric acid (pH9 with NaOH) are added to the solution and theresulting solution is heated in a 100° C. bath for 7.5 minutes. Areddish-orange colour will appear. Two ml of extracting solution(acetone and hydrochloric acid) are then added in order to stabilize thecolour. A spectrophotometric reading can now be accomplished.

Calculation of the enzymatic activity:

1 neuraminidase unit is defined as the micromols (μmol) of formed NANAper ml and per minute at 37° C., using GDia as the substrate; therefore,the activity of 1 ml of an enzymatic solution may be represented asfollows: ##EQU1## wherein OD is the optical density of NANA, as measuredby the spectrophotometer; 0.0872 is the standard OD obtained from 1 μgof NANA; 309.28 is the molecular weight of NANA and 20 is the standarddilution factor employed according to the present invention. Should afurther dilution be carried out, also this latter has to be insertedinto the equation.

The specific activity of the enzyme is defined as Unit/mg of protein.

Features of the thus Obtained Enzyme

The neuraminidase obtained by means of the process according to thepresent invention is characterized by the following features:

1) Functional action

It catalyses the reaction by which a residue of the N-acetyl-neuraminicacid is released from GDia, giving rise to the formation of GM1.

2) Substrate specificity

The enzyme tested had been previoulsy purified according to theprocedure previoulsy described.

Thr activity of the neuraminidase obtained according to the presentprocess is highly specific for the alpha configuration of GDia; howeverit will also catalyse the releasing of NANA from alpha and beta mixturesof the di- and tri-gangliosides as well as from synthetic NANA-lactose,as per the following table:

    ______________________________________                                        Substrate        Relative activity (%)                                        ______________________________________                                        GDia alpha 2-3   100                                                          GAmix alpha 2-3   60                                                          alpha 2-6                                                                     NANA-lactose alpha 2-3                                                                         100                                                          alpha 2-6                                                                     ______________________________________                                    

The neuraminidase obtained according to the present process showsnegligible activity towards colominic acid, which is known to havesialic acids linkages of the type alpha 2-8, and collocalia mucoids,which have sialic acids linkages of the type alpha 2-8 and alpha 2-6.

3) Optimum pH

The pH fop optimum activity is 5.0 at a temperature of 37° C. for 5minutes.

4) Stability

The enzyme is stabile in various organic solvents, like ethanol,methanol and butanol; it shows an increased activity in mixtures ofwater and organic solvents.

5) Range of optimum temperature (as to activity).

The enzyme is stable at a temperature from 40° to 50° C.; the optimumtemperature, as to the enzymatic activity, is 37° C.

6) Enzyme activity and inhibition.

The enzyme has a K_(m) value approximately equal to 10^(-4M) and isstrongly inhibited in the presence of NANA. The Ki fop NANA isapproximately 0.3 mM.

7) Molecular weight.

The molecular weight of the enzyme tested for substrate specificity,determined by gel permeation chromatography on Sephadex G-100 and by gelelectrophoresis (by SDS polyacrilamide electrophoresis, as described byLaemmli in "Cleavage of structural proteins during assembly of the headof bacteriophage T4", Nature, London, 227, pag. 680-685, 1970), showsthat the enzyme has a molecular weight of 55,000-60,000.

Variable amounts of the isoenzyme fraction having Molecular Weight80,000 can be occasionally present as by-product in the crude reactionsmixtures and can be separated from the main fraction having molecularweight 55,000-60,000 according to the purification procedure beforedescribed.

A neuraminidase sample, prepared according to Uchida et. al (Uchida Y.et al., J. Blochem., 82, 1425-1433, 1977) and analysed under the abovementioned electrophoresis conditions in comparison with theneuraminidase produced according to the present process, showed only aband corresponding to molecular weight 80,000.

It is worth noting that the enzyme produced by Uchida, other thanshowing a different molecular weight, also shows a different substratespecificity, being active towards colominic acid.

8) Purity.

Neuraminidase is free from toxins and from polluting proteolyticenzymes.

We claim:
 1. A process for preparing neuraminidase, comprising thefollowing steps:a) growing a culture of Arthrobacter ureafaciens ATCC7562 on a solid agar medium in the presence of N-acetyl-neuraminic acidand casein hydrolysate, to obtain a small glossy brilliant colony,herein defined as Arthrobacter Sp.; b) growing a culture of the aboveobtained Arthrobacter Sp in a liquid medium containingN-acetyl-neuraminic acid, in a concentration comprised between 0.1 and1%, and casein hydrolysate; c) once steady state conditions are reached,the culture of Arthrobacter Sp. is continued under chemostasisconditions, by feeding said culture with a fresh medium which containsN-acetyl-neuraminic acid and is free from casein hydrolysate, to produceneuraminidase; d) recovering the thus obtained neuraminidase from themedia by means of filtration followed by concentration, ultrafiltration,dialysis, and flow through an ion exchange resin.
 2. The process asclaimed in claim 1, wherein the small glossy brilliant colony ofArthrobacter Sp is isolated after 24 hours of growth on the solidmedium.
 3. The process as claimed in claim 2, wherein in step a) pH iscomprised between 7.0 and 7.5 and the temperature is comprised between25° and 30° C.
 4. The process as claimed in claim 1, wherein the cultureof Arthrobacter Sp. during steps b) and c) is effected understirring-aeration conditions, at pH values comprised between 7.0 and 7.5and at temperatures between +20° C. and +30° C.
 5. The process asclaimed in claim 1, wherein steps b) and c) are carried out at pH 7.3,and at a temperature of 26° C.
 6. The process as claimed in claim 1,wherein the concentration of N-acetyl-neuraminic acid during step b)prior to reaching steady state conditions is comprised between 0.1 and1% w/v, and the concentration of casein hydrolysate is comprised between0.1 and 0.5% w/v, and in step c) after reaching steady state conditionsthe concentration of N-acetyl-neuraminic acid during step c) varies from0.05 to 1%.
 7. The process as claimed in claim 1, wherein in step b) theArthrobacter Sp is inoculated in an amount to provide an initial opticaldensity of the cellular suspension of 0.1-0.2 Optical Density units. 8.The process as claimed in claim 1, wherein steady state conditions arereached in step b) when the optical density of the cells in reactionmedium reaches values comprised between 1.5 to 4 Optical Density units,and during step c) after reaching steady state conditions said freshmedium is fed into the reaction vessel at a rate so as to maintain theoptical density between 3 and 5 Optical Density units.
 9. The process asclaimed in claim 1, wherein the process prior to reaching steady stateconditions is carried out using as the growing medium for ArthrobacterSp. an aqueous solution containing casein hydrolysate andN-acetyl-neuraminic acid as the only carbon sources and the processafter reaching steady state conditions is carried out using during stepc) a fresh medium containing yeast extract other thanN-acetyl-neuraminic acid as the organic nutrients.
 10. The process asclaimed in claim 9, wherein prior to reaching steady state conditions,the concentrations of casein hydrolysate and N-acetyl-neuraminic acidare both 0.5% and in the process during step c) after reaching steadyconditions the N-acetyl-neuraminic acid is 0.5% and the concentration ofthe yeast extract is 0.4%; both in step b) and c) the pH is 7.3 and thetemperature is 26° C.
 11. The process as claimed in claim 9, whereinsteady state conditions are reached at the end of step b) when theoptical density of the cells in the medium is comprised between 2.0-2.5and during step c), after reachinq steady state conditions, the freshmedium is fed when the optical density fluctuates between 1.4 and 2.5,and it is pumped into the reaction vessel at a flow rate so as tomaintain the optical density between 4 and 5 Optical Density units[O.D].
 12. The process as claimed in claim 9, wherein the fresh mediumis fed at a flow rate comprised between 27 to 35 ml/min.
 13. The processas claimed in claim 1, wherein the filtration is carried out on amembrane of cellulose acetate having a pore diameter of 0.2 μm; theconcentration is effected through a cellulose acetate membrane showing amolecular cut-off of 10,000 daltons; the dyalisis is effected against apH 5.3 acetate buffer; and the ion exchange resin is a resin of dextrancross-linked with epichlorohydrin, functionalised with carboxymethylgroups and with an exclusion molecular weight limit of 2×10⁵, balancedto pH 5.3 with an acetate buffer.